Constitutive Neuronal Nitric Oxide Synthase Research Articles

Endotoxin Stimulates Nitric Oxide Production in the Paraventricular Nucleus of the Hypothalamus through Nitric Oxide Synthase I: Correlation with Hypothalamic-Pituitary-Adrenal Axis Activation

Nitric oxide (NO) is an unstable gas that is produced in brain tissues involved in the control of the activity of the hypothalamus-pituitary-adrenal (HPA) axis. Transcripts for constitutive neuronal NO synthase (NOS I), one of the enzymes responsible for NO formation in the brain, is up-regulated by systemic endotoxin[ lipopolysaccharide (LPS)] injection. However, this change is delayed compared with LPS induced-ACTH release, which makes it difficult to determine whether it is functionally important for the hormonal response. To obtain a more resolutive time course of the NO response, we first measured NO in microdialysates of the paraventricular (PVN) nucleus of the hypothalamus. The iv injection of 100 μg/kg LPS induced a rapid and short-lived increase in concentrations of this gas, which corresponded to the initiation of the ACTH response. LPS-induced Ca2+-dependent NOS activity in the PVN as well as the number of PVN cells expressing citrulline (a compound produced stoichiometrically with NO) also increased significantly over a time course that corresponded to ACTH and corticosterone release. Finally, blockade of NO production with the arginine derivative Nω-nitro-l-arginine-methylester (L-NAME; 50 mg/kg, sc), which attenuated the ACTH response to LPS, virtually abolished basal NOS activity in the PVN, as well as anterior and neurointermediate lobes of the pituitary, and prevented the appearance of citrulline in the PVN of rats injected with LPS. Collectively, these results show that LPS-induced activation of the HPA axis correlates with the activation of the PVN NOergic system, and supports a stimulatory role for NO in the modulation of the HPA axis in response to immune challenges.

Cilostazol, a cyclic AMP phosphodiesterase inhibitor, stimulates nitric oxide production and sodium potassium adenosine triphosphatase activity in SH-SY5Y human neuroblastoma cells

Deficiencies in cellular cyclic AMP (cAMP) and nitric oxide (NO) production are thought to be involved in the pathogenesis of diabetic neuropathy. We used a human neuroblastoma cell line, SH-SY5Y, to investigate the effect of cilostazol, a specific cAMP phosphodiesterase inhibitor, on NO production and Na +, K +-ATPase activity. SH-SY5Y cells were cultured under 5 or 50 mM glucose for 5–6 days, the cells were then exposed to cilostazol or other chemicals and nitrite, cAMP and Na +, K +-ATPase activity were measured. In cells grown in 50 mM glucose, cilostazol was observed to increase significantly both NO production and cellular cAMP accumulation in a time- and dose-dependent manner. Cilostazol also significantly recovered reduced levels of protein kinase A activity (PKA) in 50 mM glucose. Furthermore, a PKA inhibitor, H-89 significantly suppressed the increase in NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production by activating PKA. Cilostazol did not affect either sorbitol or myo-inositol concentrations. Dexamethazone, which is known to induce inducible NO synthase, had no effect on NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production catalyzed by neuronal constitutive NO synthase (ncNOS) in SH-SY5Y cells. L-arginine, which is an NO agonist enhanced Na +, K +-ATPase activity in cells grown in 50 mM glucose, NG-nitro-L-arginine methyl ester (L-NAME), which is an NOS inhibitor inhibited basal Na +, K +-ATPase activity in 5 mM glucose and suppressed the increased enzyme activity induced by cilostazol in 50 mM glucose. The above results confirmed our previous observation that NO regulates Na +, K +-ATPase activity in SH-SY5Y cells and suggest that cilostazol increases Na +, K +-ATPase activity, at least in part, by stimulating NO production. The present results also suggest that cilostazol has a beneficial effect on diabetic neuropathy by improving Na +, K +-ATPase activity via directly increasing cAMP and NO production in nerves.

Characterization of Mouse nNOS2, a Natural Variant of Neuronal Nitric-oxide Synthase Produced in the Central Nervous System by Selective Alternative Splicing

Mouse neuronal nitric-oxide synthase 2 (nNOS2) is a unique natural variant of constitutive neuronal nitric-oxide synthase (nNOS) specifically expressed in the central nervous system having a 105-amino acid deletion in the heme-binding domain as a result of in-frame mutation by specific alternative splicing. The mouse nNOS2 cDNA gene was heterologously expressed in Escherichia coli, and the resultant product was characterized spectroscopically in detail. Purified recombinant nNOS2 contained heme but showed no L-arginine- and NADPH-dependent citrulline-forming activity in the presence of Ca2+-promoted calmodulin, elicited a sharp electron paramagnetic resonance (EPR) signal at g = 6.0 indicating the presence of a high spin ferriheme as isolated and showed a peak at around 420 nm in the CO difference spectrum, instead of a 443-nm peak detected with the recombinant wild-type nNOS1 enzyme. Thus, although the heme domain of nNOS2 is capable of binding heme, the heme coordination geometry is highly abnormal in that it probably has a proximal non-cysteine thiolate ligand both in the ferric and ferrous states. Moreover, negligible spectral perturbation of the nNOS2 ferriheme was detected upon addition of either L-arginine or imidazole. These provide a possible rational explanation for the inability of nNOS2 to catalyze the cytochrome P450-type monooxygenase reaction.

Inactivation of nitric oxide synthases and cellular nitric oxide formation by N6-iminoethyl- l-lysine and N5-iminoethyl- l-ornithine

The kinetics of inactivation of affinity-purified nitric oxide synthase isoforms by N 6-iminoethyl- l-lysine (NIL) and N 5-iminoethyl- l-ornithine (NIO) has been examined. Each of the agents produced a time and concentration dependent first order inactivation of the nitric oxide synthase isoforms that required exposure of the NO synthase to drug under conditions that supported catalysis, consistent with the proposal that these agents act as alternate substrate, mechanism-based inactivators. As measured at 100 μM arginine, NIL and NIO were equally efficient as inactivators of the cytokine-inducible nitric oxide synthase exhibiting apparent second order inactivation rate constants of 31.5 and 32.0 mM −1 min −1 respectively. By contrast, NIL and NIO were less efficient as inactivators of the constitutive neuronal nitric oxide synthase isoform exhibiting apparent second order inactivation rate constants of 0.79 and 8.4 mM −1 min −1 respectively. As measured at 100 μM extracellular arginine, NIL and NIO produced a time and concentration dependent inactivation of the NO synthetic capability of cytokine-induced murine macrophage RAW 264.7 cells exhibiting apparent second order inactivation rate constants of 3.1 and 1.8 mM −1 min −1. The inactivated RAW cell NO synthetic capability was restored to 30% of its pretreatment value over a 3-h period despite the presence of cycloheximide.

5-Hydroxyindoles compounds and nitric oxide voltammetric detection in the rat brain: changes occurring throughout the sleep-wake cycle.

The release of serotonin may occur throughout the sleep-wake cycle according to 2 different modalities: - by the axonal nerve endings during waking; - by the dendrites and/or the soma of the nucleus raphe dorsalis (nRD) during sleep. Neuronal nitric oxide (NO), synthesised by constitutive NO synthase (NOS), is colocalized with neurotransmitters such as GABA, acetylcholine, somatostatin, serotonin, etc. In order to evaluate its modalities of release throughout the rat sleep-wake cycle, a sensor allowing its specific detection in freely moving animals was prepared. In the cortex, the highest NO signal occurs during the waking state (W=100%) versus slow wave sleep (SWS=-6%) and paradoxical sleep (PS=-9%). The mild variations observed might reflect a mean of the individual sleep-wake cycle variations attached to each NO source (GABAergic interneurons, cholinergic and serotoninergic axonal nerve endings, etc.).

Lower than normal expression of brain nitric oxide synthase gene in the hypothalamus of deoxycorticosterone acetate-salt hypertensive rats.

To elucidate the role of brain nitric oxide produced by neuronal constitutive nitric oxide synthase in sodium-induced hypertension. Diets containing a high (8% NaCl), a medium (2% NaCl), and a low (0.2% NaCl) sodium content were administered to Wistar rats aged 12 weeks for 10 days or 8 weeks until they were killed. Male Wistar rats administered either deoxycorticosterone acetate, 1% NaCl or both and the respective controls were killed 2 weeks (during prehypertensive stage) or 6 weeks (during hypertensive stage) after the start of treatment. The hypothalamus and lower brainstem were excised for extraction of total RNA. Reverse transcription polymerase chain reactions of constitutive nitric oxide synthase messenger RNA and glyceraldehyde-3-phosphate dehydrogenase messenger RNA were performed, and constitutive nitric oxide synthase messenger RNA levels were expressed relative to glyceraldehyde-3-phosphate dehydrogenase messenger RNA levels. A high sodium intake for 10 days tended to decrease constitutive nitric oxide synthase messenger RNA levels in the hypothalamus, compared with effect of a low sodium intake. Constitutive nitric oxide synthase messenger RNA levels in the hypothalamus of deoxycorticosterone acetate-salt hypertensive rats were lower than those in the control sham-operated rats. Neither alteration of sodium intake nor administration of deoxycorticosterone with and without sodium affected constitutive nitric oxide synthase gene expression in the lower brainstem. Expression of neuronal constitutive nitric oxide synthase gene is downregulated in the hypothalamus of deoxycorticosterone acetate-salt hypertensive rats. This lower than normal expression of neuronal constitutive nitric oxide synthase gene in the hypothalamus could be an adaptive response to sodium-induced hypertension, and suggests that nitric oxide produced by hypothalamic constitutive nitric oxide synthase plays a role in maintenance of blood pressure in relation to sodium balance in rats.

Cutting Edge: Antisense Knockdown of Inducible Nitric Oxide Synthase Inhibits Induction of Experimental Autoimmune Encephalomyelitis in SJL/J Mice

Abstract We used an antisense oligodeoxynucleotide (ODN) complementary to inducible nitric oxide synthase (iNOS) to inhibit experimental autoimmune encephalomyelitis (EAE) in female SJL/J mice, an animal model for multiple sclerosis. The antisense ODN was administered intraventricularly to mice daily for 10 days beginning at the time of adoptive transfer of myelin basic protein-specific T lymphocytes. The antisense ODN treatment significantly reduced the clinical score of EAE and blocked iNOS mRNA and protein synthesis, as well as iNOS enzyme activity within the central nervous system. The levels of nitric oxide and cyclic guanosine monophosphate were also significantly reduced by the antisense ODN treatment. Neither sense nor random ODN affected clinical EAE or iNOS expression. Moreover, the protein and enzyme activity level of constitutive neuronal nitric oxide synthase was not affected by the antisense ODN. Thus, we have shown that the iNOS antisense ODN specifically blocked iNOS expression and ameliorated the induction of EAE.

Spinal and supraspinal antinociceptive action of dipyrone in formalin, capsaicin and glutamate tests. Study of the mechanism of action

Dipyrone injected intraperitoneally (i.p.) or subplantarly into the mouse paw caused dose-related antinociception against the early and the late phases of formalin-induced licking, with mean ID 50 values of 154.5 and 263.7 μmol/kg, and 2.6 and 1.2 μmol/paw, respectively. Given either by intracerebroventricular (i.c.v.) or by intrathecal (i.t.) routes, dipyrone produced a similar inhibition of both phases of the formalin-induced licking, with mean ID 50 values of 0.4 and 1.3 μmol/site, and 0.4 and 0.9 μmol/site against the early and the late phase of the formalin response, respectively. Dipyrone, given by i.p., subplantar, i.t. or i.c.v. routes, caused dose-related antinociception of capsaicin-induced licking. The mean ID 50 values were: 207.6 μmol/kg, 2.2 μmol/paw, 0.4 μmol/site and 0.14 μmol/site, respectively. In addition, dipyrone given i.p. caused a significant increase of the latency both in the hot-plate and the tail-flick assays. Dipyrone, given i.p., i.t. or i.c.v., reversed significantly the hyperalgesia caused by i.t. injection of glutamate, with mean ID 50 values of 9 μmol/kg, 29 nmol/site and 94 nmol/site, respectively. The antinociception caused by dipyrone was not influenced by naloxone, l-arginine, phaclofen, glibenclamide, p-chlorophenylalanine methyl ester, pertussis toxin or by adrenal gland hormones, when assessed against the formalin assay. Dipyrone analgesic action was not secondary to its anti-inflammatory effect, nor was it associated with non-specific effects such as muscle relaxation or sedation actions of animals. Dipyrone at a higher concentration caused significant inhibition of [ 3H]glutamate binding (37%) in cerebral cortical membranes from both mice and rats. However, dipyrone had no significant effect on brain constitutive neuronal nitric oxide synthase activity. It is concluded that dipyrone produces peripheral, spinal and supraspinal antinociception when assessed on formalin and capsaicin-induced pain as well as in glutamate-induced hyperalgesia in mice. Dipyrone antinociception seems unlikely to involve an interaction with the l-arginine-nitric oxide pathway, serotonin system, activation of G i protein sensitive to pertussis toxin, interaction of ATP-sensitive K + channels, GABA B receptors, or the release of endogenous glucocorticoids. However, a modulatory effect on glutamate-induced hyperalgesia and, to a lesser extent, an interaction with glutamate binding sites, seems to account for its analgesic action.

Increased neuronal nitric oxide synthase expression in brain following portacaval anastomosis

It has previously been suggested that increases of l-arginine uptake into brain following portacaval shunting may result in increased activities of constitutive neuronal nitric oxide synthase (nNOS). In order to further address this issue, nNOS protein and gene expression were studied by Western blot analysis using a monoclonal nNOS antibody and RT-PCR respectively in the brains of rats following portacaval shunting or sham operation. Portacaval shunting resulted in a 2-fold increase ( P<0.01) in nNOS protein and a concomitant 2.4-fold increase ( P<0.01) in nNOS mRNA. Increased nNOS activity in brain and the resulting increase in nitric oxide production could contribute to the increased cerebral blood flow and to the pathogenesis of hepatic encephalopathy in chronic liver disease.

Association analysis of TG repeat polymorphism of the neuronal nitric oxide synthase gene with essential hypertension.

The nitric oxide synthase (NOS) gene is thought to be involved in essential hypertension because nitric oxide is implicated in endothelium-mediated or nitroxidergic neuron-mediated vasodilation. Using simple tandem repeat DNA polymorphism of the neuronal constitutive NOS (nNOS) gene, we carried out an association study in patients with essential hypertension. One hundred and thirty-one patients with essential hypertension and 147 subjects with normal blood pressure were studied. Polymerase chain reaction was applied to amplify the TG repeat site in the nNOS gene, and alleles based on the TG repeat number were determined. Eight alleles were identified in this study of Japanese subjects. Overall distributions of allele frequencies in the two groups were not significantly different. Thus, the genes detected by examination of this microsatellite polymorphism in the nNOS gene are not associated with essential hypertension.

Substrate Binding and Calmodulin Binding to Endothelial Nitric Oxide Synthase Coregulate Its Enzymatic Activity

Endothelial nitric oxide synthase (NOS) is a constitutively expressed flavin-containing heme protein that catalyzes the formation of NO froml-arginine, NADPH, and molecular oxygen. We purified bovine endothelial NOS from transfected embryonic kidney cells by conventional chromatographic techniques and characterized the activity of the detergent-solubilized enzyme. Endothelial NOS displays a much lower specific activity of NO synthesis (143 ± 11 nmol NO/min/mg enzyme) than the constitutive neuronal NOS or inducible NOS isoforms. Like the neuronal isoform, endothelial NOS requires binding of Ca2+/calmodulin to achieveVmaxNO synthase activity; however, we observed a basal level of NO synthesis even when Ca2+/calmodulin was omitted and 0.5 mmEDTA was present in the assay solution. Moreover, endothelial NOS demonstrates a high-affinity bonding interaction with calmodulin such that the enzyme as purified has a NO synthase activity at about 80% ofVmax. We also observed a more than twofold increase in NADPH consumption by endothelial NOS when it was coupled to arginine oxygenation as opposed to when oxygen is activated in the absence of substrate. Substrate binding was also shown to stimulate heme reduction in the absence of added calmodulin. Thus, the enzymatic synthesis of NO froml-arginine by endothelial NOS appears to be partially regulated by binding of both calmodulin and substrate. These findings for endothelial NOS represent a significant departure from the enzymatic properties of the other constitutive NOS isoform, neuronal NOS, and we interpret this result in terms of the physiological implications.

Upregulation of endothelial and neuronal constitutive nitric oxide synthase in pregnant rats.

Pregnancy is characterized by hemodynamic and body fluid alterations. Increased nitric oxide (NO) production has been suggested to play a role in the hemodynamic alterations of pregnancy and has also been reported to increase arginine vasopressin (AVP) release. We therefore hypothesized that gestation could increase both NO synthase (NOS) constitutive isoforms, neuronal NOS and endothelial NOS, and thereby contribute to the hyposmolality and peripheral arterial vasodilation of pregnancy, respectively. The present study was therefore undertaken to examine the constitutive NOS isoforms in aortas, mesenteric arteries, and hypothalami of pregnant rats on day 20 of gestation compared with age-matched nonpregnant rats. Plasma AVP was determined by radioimmunoassay and hypothalamic mRNA AVP by solution hybridization assay. Hypothalamic neuronal NOS was assessed by Northern blot and Western blot; endothelial NOS was assessed by Western blot in arteries and hypothalamus. The results demonstrated that 1) plasma AVP and hypothalamic AVP mRNA are increased in pregnant rats (n = 8), 2) neuronal NOS protein and mRNA are increased in hypothalamus of pregnant rats (n = 4), and 3) endothelial NOS expression, as assessed by Western blot analysis, is increased in both conductance (aorta) as well as resistance (mesenteric) arteries of pregnant rats (n = 4). We conclude that both of the constitutive NOS isoforms are increased in pregnant rats, suggesting that the peripheral arterial vasodilation and hyposmolality of pregnancy could be mediated by these isoforms.

Neuronal constitutive nitric oxide synthase is involved in murine enteric inhibitory neurotransmission.

Mice lacking neuronal nitric oxide synthase gene (ncNOS) were used to determine the enzymatic source of nitric oxide (NO) and its relationship with other putative inhibitory neurotransmitters. Inhibitory junction potentials (IJP) of circular smooth muscle of gastric fundus were studied. The IJP in the wild-type mice consists of overlapping components, the fast and slow IJPs. NOS inhibitor L-NA or VIP receptor antagonist VIP(10-28), blocks the slow IJP but not the fast IJP. The fast UP is blocked by alpha-beta methylene ATP tachyphylaxis, by reactive blue 2, and by apamin. The IJP in the ncNOS-deficient [ncNOS(-)] mutant is of short duration and is abolished by blockers of the fast IJP, but is unaffected by blockers of the slow UP. Exogenous VIP produces membrane hyperpolarization in strips from wild-type but not ncNOS(-) mice. The hyperpolarizing action of VIP is resistant to nifedipine but is sensitive to omega-conotoxin GVIA. (a) NO derived from ncNOS is an inhibitory neurotransmitter rather than a postjunctional mediator; (b) VIP is a prejunctional neurotransmitter that causes release of evanescent NO; and (c) ATP acts in parallel with the VIP/NO pathway.

Nitric oxide synthase in cat brain: Cofactors—enzyme-substrate interaction

Nitric oxide, derived from l-arginine by the enzyme nitric oxide synthase, is an activator of the soluble guanylate cyclase and a cellular messenger. This work demonstrates that, in cat brain, the neuronal constitutive nitric oxide synthase activity is a) NADPH/calcium dependent, b) independent upon exogenous calmodulin in crude brain supernatant, c) significantly enhanced by exogenous FAD and tetrahydrobiopterin ( V max: 118 instead of 59.4 pmol of citrulline formed ·mg of prot· −1 min −1, d) inhibited by calcium chelators and calmodulin antagonist, and e) present in several neuroanatomical structures. Moreover, the K m value for l-arginine was of 11 μM instead of 41 μM in the presence of FAD and tetrahydrobiopterin in the incubation mixture, thus demonstrating that these cofactors are able to stabilize the enzyme-substrate interactions.

Expression of inducible nitric oxide synthase in smooth muscle cells from rat penile corpora cavernosa.

Nitric oxide (NO), the main mediator of penile erection, is assumed to be synthesized in the penis by the neuronal constitutive nitric oxide synthase (nNOS). However, nNOS has not been identified in the penile smooth muscle, the target of NO action. The other NOS isozymes, the inducible NOS (iNOS) and the endothelial NOS (eNOS) have not been reported in any penile tissue. The smooth muscle vascular and trabecular tissue from rat corpora cavernosa is represented in vitro by cell cultures designated RPSMC. To determine whether iNOS can be expressed in penile smooth muscle, RPSMC were treated with different lymphokines and/or bacterial lipopolysaccharide (LPS). The selected inducer, LPS/interferon, elicited at 48 hours up to a 50-fold increase in nitrites in the medium; the nitroarginine methyl ester (L-NAME), aminoguanidine, actinomycin D, cycloheximide, transforming growth factor-beta1 (TGF-beta1), and dexamethasone, but was resistant to nifedipine and platelet-derived growth factor AB (PDGF-AB). iNOS induction increased with cell passage. The [3H]L-arginine/citrulline measurement of NO synthesis with intact cells confirmed these results. Incubations of soluble and particulate fractions showed that the cytosol contained most of the activity (Km = 43 microM), which was partially inhibited by ethyleneglycal-bis-tetraacetic acid (EGTA). The 4.4-kb iNOS mRNA peaked at a late period (24-30 hours) and remained high for up to 72 hours. iNOS mRNA induction was strongly inhibited by actinomycin D and dexamethasone, partially inhibited by TGF-beta1, inhibited slightly by PDGF-AB, and unaffected by nifedipine. These results show that iNOS can be expressed in RPSMC in a cell passage-dependent fashion that has so far not been reported for other cell lines, and that the induction reaches much higher levels than in rat or human vascular smooth muscle cells. The expression pattern is also distinctive for the penile cells in time course of induction, Ca2+ dependence, response to certain agents, and mRNA stability.

In the Rat Pineal Gland, but Not in the Suprachiasmatic Nucleus, the Amount of Constitutive Neuronal Nitric Oxide Synthase Is Regulated by Environmental Lighting Conditions

To investigate whether the expression of neuronal isoform of nitric oxide synthase is photoneurally regulated, we examined the amount of the enzyme by means of Western Blot analysis under different environmental lighting conditions in two photoneuronally regulated tissues, the pineal gland and the suprachiasmatic nucleus of the rat. In the pineal gland nitric oxide synthase immunoreactivity was strikingly decreased when rats had been exposed for 8 days to light:dark 20:4 conditions or to constant light. The decline in nitric oxide synthase immunoreactivity following LL reversed after 4 days under light:dark 12:12. We conclude that in the rat pineal the amount of neuronal nitric oxide synthase is controlled by environmental lighting conditions. That it is not justified to extrapolate from the pineal to other photoneuronally regulated centers is illustrated by the present finding that the suprachiasmatic nucleus did not reveal changes in the amount of neuronal nitric oxide synthase.

Inhibition of constitutive nitric oxide synthase in the brain by pentamidine, a calmodulin antagonist

Nitric oxide (NO) which is produced by activation of Ca 2+/calmodulin-dependent NO synthase is known to induce neuronal damage. We examined the effects of 3′-azido-2′,3′-dideoxythymidine (AZT, a reverse transcriptase inhibitor), pentamidine (a therapeutic drug for Pneumocystis carinii pneumonia) and calmodulin antagonists such as trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on NO synthase activation. Although AZT had no effect on the activity of constitutive neuronal NO synthase, pentamidine inhibited the activation of neuronal NO synthase as did trifluoperazine and W-7. The inhibition by pentamidine was prevented by the addition of purified calmodulin. In addition, pentamidine inhibited calmodulin-dependent activation of neuronal NO synthase purified from rat cerebellum. From these results, it is suggested that pentamidine inhibits the neuronal NO synthase activation by probably acting as a calmodulin antagonist.

Effect of neurotropic virus infection on neuronal and inducible nitric oxide synthase activity in rat brain.

To elucidate the potential role of inducible nitric oxide synthase (iNOS) and neuronal constitutive nitric oxide synthase (cNOS) in the pathogenesis of virus-induced encephalopathy, the activities of both NOS isoforms were determined in the brains of rats infected with Borna disease virus (BDV) or rabies virus. iNOS activity strongly increased, whereas neuronal cNOS activity significantly decreased in a time-dependent manner after either BDV or rabies virus infection. Choline acetyltransferase activity in the brain remained unchanged during both virus infections, suggesting that the decrease in cNOS activity does not reflect a generalized neuronal loss. Immunohistochemistry and Northern blot analyses indicate that the decrease in neuronal cNOS activity is due to a decrease in cNOS protein and mRNA synthesis. These results suggest that both an excessive generation of NO by activated macrophages or microglia, as well as a decrease of NO production in neurons may contribute to the neuropathogenesis of neurotropic virus infections.

Immunohistochemical demonstration of a paracrine role of nitric oxide in bronchial function.

We addressed the controversial role of nitric oxide (NO) in bronchial function by an immunohistochemical study of the localization of NO synthase (NOS) and its effector protein, soluble guanylate cyclase, in rat bronchus. For this study, a monoclonal antibody to the bovine constitutive neuronal NOS was developed and characterized. In Western blot analysis, this monoclonal antibody (anti-NOS antibody) reacted with bovine cerebellum NOS (150 kDa) as well as with structurally different NOSs from cultured bovine aortic endothelial cells (130 kDa) and cultured RAW 264.7 macrophages (130 kDa). The reactivity of anti-NOS antibody was confirmed by immunohistochemical staining of rat cerebellum, arterial endothelial cells, and cultured stimulated macrophages. When the distribution of NOS in rat airway was characterized, the anti-NOS antibody showed immunoreactivity within respiratory epithelium but not in the bronchial smooth muscle. The NADPH-diaphorase staining correlated with the immunostaining. In contrast, a monoclonal antibody to the rat lung-soluble guanylate cyclase immunostained respiratory smooth muscle but not epithelium. This study suggests a paracrine role for NO in bronchial function analogous to the function of the NOS-soluble guanylate cyclase pathway in blood vessels.

Biphasic Effect of Estrogen on Neuronal Constitutive Nitric Oxide Synthase via Ca2+-Calmodulin Dependent Mechanism

Estrogen is known to retard the development of atherosclerosis and to work in the brain, but the mechanism of hormonal action is completely unknown. We investigated the effect of estrogen on the activity of neuronal constitutive nitric oxide synthase (NNOS). A low concentration of estrogen (10−10-7 M) enhanced the activity of homogenates of the cytosol fraction of rabbit cerebellums and also that of partially purified NNOS, and high dose (10−6-5 M) attenuated them. The study using estrogen receptor antagonists, tamoxifen, clomiphene, and ICI182780 suggested that estrogen receptor did not relate significantly to those effects of 17β-estradiol. 17α-estradiol or progesterone did not change significantly it in low doses, although moderately inhibited it in high doses. Estrogen enhanced the fluorescence of dansyl-calmodulin in low doses and attenuated it in high doses, suggesting that estrogen affects Ca2+-calmodulin directly. This study demonstrated that estrogen has a biphasic effect on the activity of NNOS through a Ca2+-calmodulin.